Amy B. Rosen

Finding Fluorescent Needles in the Cardiac Haystack: Tracking Human Mesenchymal Stem Cells Labeled with Quantum Dots for Quantitative In Vivo Three‐Dimensional Fluorescence Analysis

Stem cells show promise for repair of damaged cardiac tissue. Little is known with certainty, however, about the distribution of these cells once introduced in vivo. Previous attempts at tracking delivered stem cells have been hampered by the autofluorescence of host tissue and limitations of existing labeling techniques. We have developed a novel loading approach to stably label human mesenchymal stem cells with quantum dot (QD) nanoparticles. We report the optimization and validation of this long‐term tracking technique and highlight several important biological applications by delivering labeled cells to the mammalian heart. The bright QD crystals illuminate exogenous stem cells in histologic sections for at least 8 weeks following delivery and permit, for the first time, the complete three‐dimensional reconstruction of the locations of all stem cells following injection into the heart.

Xenografted Adult Human Mesenchymal Stem Cells Provide a Platform for Sustained Biological Pacemaker Function in Canine Heart

Background— Biological pacemaking has been performed with viral vectors, human embryonic stem cells, and adult human mesenchymal stem cells (hMSCs) as delivery systems. Only with human embryonic stem cells are data available regarding stability for >2 to 3 weeks, and here, immunosuppression has been used to facilitate survival of xenografts. The purpose of the present study was to determine whether hMSCs provide stable impulse initiation over 6 weeks without the use of immunosuppression, the “dose” of hMSCs that ensures function over this period, and the catecholamine responsiveness of hMSC-packaged pacemakers. Methods and Results— A full-length mHCN2 cDNA subcloned in a pIRES2-EGFP vector was electroporated into hMSCs. Transfection efficiency was estimated by GFP expression. IHCN2 was measured with patch clamp, and cells were administered into the left ventricular anterior wall of adult dogs in complete heart block and with backup electronic pacemakers. Studies encompassed 6 weeks. IHCN2 for all cells was 32.1±1.3 pA/pF (mean±SE) at −150 mV. Pacemaker function in intact dogs required 10 to 12 days to fully stabilize and persisted consistently through day 42 in dogs receiving ≥700 000 hMSCs (≈40% of which carried current). Rhythms were catecholamine responsive. Tissues from animals killed at 42 days manifested neither apoptosis nor humoral or cellular rejection. Conclusions— hMSCs provide a means for administering catecholamine-responsive biological pacemakers that function stably for 6 weeks and manifest no cellular or humoral rejection at that time. Cell doses >700 000 are sufficient for pacemaking when administered to left ventricular myocardium.

Mapping Mechanical Strain of an Endogenous Cytoskeletal Network in Living Endothelial Cells

A central aspect of cellular mechanochemical signaling is a change of cytoskeletal tension upon the imposition of exogenous forces. Here we report measurements of the spatiotemporal distribution of mechanical strain in the intermediate filament cytoskeleton of endothelial cells computed from the relative displacement of endogenous green fluorescent protein (GFP)-vimentin before and after onset of shear stress. Quantitative image analysis permitted computation of the principal values and orientations of Lagrangian strain from 3-D high-resolution fluorescence intensity distributions that described intermediate filament positions. Spatially localized peaks in intermediate filament strain were repositioned after onset of shear stress. The orientation of principal strain indicated that mechanical stretching was induced across cell boundaries. This novel approach for intracellular strain mapping using an endogenous reporter demonstrates force transfer from the lumenal surface throughout the cell.

Enhanced recovery of mechanical function in the canine heart by seeding an extracellular matrix patch with mesenchymal stem cells committed to a cardiac lineage.

The need to regenerate tissue is paramount, especially for the heart that lacks the ability to regenerate after injury. The urinary bladder extracellular matrix (ECM), when used to repair a right ventricular defect, successfully regenerated some mechanical function. The objective of the current study was to determine whether the regenerative effect of ECM could be improved by seeding the patch with human mesenchymal stem cells (hMSCs) enhanced to differentiate down a cardiac linage. hMSCs were used to form three-dimensional spheroids. The expression of cardiac proteins was determined in cells exposed to the spheroid formation and compared with nonmanipulated hMSCs. To determine whether functional calcium channels were present, the cells were patch clamped. To evaluate the ability of these cells to regenerate mechanical function, the spheroids were seeded on ECM and then implanted into the canine heart to repair a full-thickness right ventricular defect. As a result, many of the cells spreading from the spheroids expressed cardiac-specific proteins, including sarcomeric alpha-actinin, cardiotin, and atrial natriuretic peptide, as well as the cell cycle markers cyclin D1 and proliferating cell nuclear antigen. A calcium current similar in amplitude to that of ventricular myocytes was present in 16% of the cells. The cardiogenic cell-seeded scaffolds increased the regional mechanical function in the canine heart compared with the unmanipulated hMSC-seeded scaffolds. In addition, the cells prelabeled with fluorescent markers demonstrated myocyte-specific actinin staining with sarcomere spacing similar to that of normal myocytes. In conclusion, the spheroid-derived cells express cardiac-specific proteins and demonstrate a calcium current similar to adult ventricular myocytes. When these cells are implanted into the canine heart, some of these cells appear striated and mechanical function is improved compared with the unmanipulated hMSCs. Further investigation will be required to determine whether the increased mechanical function is due to a differentiation of the cardiogenic cells to myocytes or to other effects.

Increased Myocyte Content and Mechanical Function Within a Tissue-Engineered Myocardial Patch Following Implantation

During the past few years, studies involving the implantation of stem cells, chemical factors, and scaffolds have demonstrated the ability to augment the mammalian hearts native regenerative capacity. Scaffolds comprised of extracellular matrix (ECM) have been used to repair myocardial defects. These scaffolds become populated with myocytes and provide regional contractile function, but quantification of the myocyte population has not yet been conducted. The purpose of this study was to quantitate the myocyte content within the ECM bioscaffold and to correlate this cell population with the regional mechanical function over time. Xenogenic ECM scaffolds derived from porcine urinary bladder were implanted into a full-thickness, surgically induced, right ventricular-free wall defect in a dog model. Zero, 2, and 8 weeks following implantation, regional function and myocyte content were determined in each patch region. Regional function did not significantly increase from 0 to 2 weeks. At 8 weeks, however, regional stroke work increased to 3.7 +/- 0.7% and systolic contraction increased to 4.4 +/- 1.2%. The myocyte content also significantly increased during that period generating a linear relationship between regional function and myocyte content. In conclusion, ECM used as a myocardial patch increases both the regional function and the myocyte content over time. The mechanical function generated in the patch region is correlated with the quantity of local tissue myocytes.

Regenerative therapies in electrophysiology and pacing

The prevention and treatment of cardiac arrhythmias conferring major morbidity and mortality is far from optimal, and relies heavily on devices and drugs for the partial successes that have been seen. The greatest success has been in the use of electronic pacemakers to drive the hearts of patients having high degree heart block. Recent years have seen the beginnings of attempts to use novel approaches available through gene and cell therapies to treat both brady- and tachyarrhythmias. By far the most successful approaches to date have been seen in the development of biological pacemakers. However, the far more difficult problems posed by atrial fibrillation and ventricular tachycardia are now being addressed. In the following pages we review the approaches now in progress as well as the specific methodologic demands that must be met if these therapies are to be successful.

Flow-induced strain focusing in the endothelial cytoskeleton

Displacement or deformation of the endothelial cell cytoskeleton by hemodynamic shear stress may reflect redistribution of intracellular force to molecular signaling complexes that initiate adaptive mechanisms. In these studies, cytoskeletal movement in response to onset of shear stress was measured in endothelial cells expressing GFP-vimentin or GFP-actin. High-resolution optical sectioning fluorescence microscopy and quantitative image processing revealed increased intermediate filament displacement with height in the cell and in downstream regions of the cytoplasm. The Lagrangian strain tensor demonstrated highly localized deformation near the coverslip. Variability in principal strain orientation increased significantly after onset of shear stress. In contrast to the spatially heterogeneous distribution of displacement and strain in the intermediate filament network, onset of shear stress induced rapid extension of actin ruffles in the upstream direction, and lamellipodia along downstream edges of the cell were often retracted. This directional response often subsided within 10-15 min and occurred in cells in confluent but not subconfluent monolayers. In addition, microfilaments in central regions of the cytoplasm were often displaced by flow onset. These data suggest that endothelial cells sense changes in shear stress and rapidly mobilize an active cytoskeletal response that may contribute to initiation of adaptive mechanotransduction networks.